1. Field of the Invention
The invention relates to a target cell stimulating peptide. The invention is also directed to a method of making the target cell stimulating peptide. Further, the invention is directed to a method of using the target cell stimulating peptide as a chemoattractant.
2. General Background and State of the Art
Neutrophils play a key role in innate immune responses. Diverse extracellular agonists modulate neutrophil function by stimulating the activities of intracellular enzymes (Robson et al. J. Immunol. 2001. 167: 1028-1038; M'Rabet et al. J. Biol. Chem. 1999. 274: 21847-21852). Recently, many reports have demonstrated the critical involvement of phospholipases in neutrophil immune response (Gijon et al. J. Leukoc. Biol. 1999. 65: 330-336; Wu et al. J. Cell Sci. 2000. 113: 2935-2940; Liscovitch et al. Biochem. J. 2000. 345: 401-415). Among these phospholipases, phospholipase A2 (PLA2) is an important enzyme that mediates several immune responses. PLA2 hydrolyzes the fatty acyl group from the sn-2 position of phospholipid and concomitantly generates lysophospholipid (Gijon et al. J. Leukoc. Biol. 1999. 65: 330-336; Puri et al. Int. J Biochem. Cell Biol. 1998. 30: 1107-1122). Arachidonic acid (AA), the product of PLA2 activity, has been implicated in the regulation of various cellular responses, including calcium influx and superoxide generation in phagocytic cells (Murthy et al. J. Biol. Chem. 1998. 273: 34519-34526; Robinson et al. Biochem. J. 1998. 336: 611-617).
Mammalian cells contain several isozymes of PLA2, namely, cytosolic PLA2 (cPLA2), calcium-independent PLA2, and secretory PLA2 (Gijon et al. J. Leukoc. Biol. 1999. 65: 330-336; Farooqui et al. J. Neurochem. 1997. 69: 889-901). Among the PLA2 isozymes, cPLA2 is regarded to play an important role in agonist-induced AA release and in the regulation of lysophospholipid levels in cells (Gijon et al. J. Leukoc. Biol 1999. 65: 330-336). Recently Dana et al. developed cPLA2-deficient mice and confirmed the role of cPLA2 in their eicosanoid production (Dana et al. J. Biol. Chem. 1998. 273: 441-445). Set against this background, cPLA2 is considered to be an important pharmacological target for several physiological responses. With this role of PLA2 in mind, particularly with respect to neutrophil function, we undertook to identify new ligands that modulate PLA2 activity, and the characterization of their action mechanisms.
Several recent studies have reported the use of combinatorial peptide libraries to identify sequences involved in various biological responses (Boen et al. J. Immunol. 2000. 165: 2040-2047; Wilson et al. J. Immunol. 1999. 163: 6424-6434; Hiemstra et al. J. Immunol 1998. 161: 4078-4082). An easy and powerful method for identifying peptide sequences in certain biological reactions was developed by Houghten et al. (Dooley et al. Methods Mol. Biol. 1998. 87: 13-24). This method, which uses a positional scanning synthetic peptide combinatorial library (PS-SPCL), has been used for various purposes, including the identification of human immunodeficiency virus protease inhibitors, interleukin-8-specific antagonists, the inhibitor for the nuclear factor of activated T cells, and the ligands of opioid receptors, and peptides responsible for modulating leukocytic cell activity (Owens et al. Biochem. Biophys. Res. Commun. 1991. 181: 402-408; Hayashi et al. J. Immunol. 1995. 154: 814-824; Aramburu et al. Science. 1999. 285: 2129-2133; Dooley et al. J. Biol. Chem. 1998. 273: 18848-18856; Baek et al. J. Biol. Chem. 1996. 271: 8170-8175).
In the present invention, we adopted the PS-SPCL method to identify the peptides that are responsible for AA release in neutrophil-like differentiated HL60 (dHL60) cells. We found 24 peptides that stimulate AA release in dHL60 cells, and found that these peptides act as chemoattractants for human phagocytes. Conversely, on the topic of the receptors of these peptides, we found that several peptides bound to the formyl peptide receptor like 1 (FPRL1). Some of the peptides were also found to bind to other receptor(s) expressed in HL60 cells. In addition, each peptide was found to be capable of stimulating shared and distinct intracellular signaling pathways.